OPTIMIZED SMALL GUIDE RNAS AND METHODS OF USE

• US 20160289673A1
• Filed: 09/29/2014
• Published: 10/06/2016
• Est. Priority Date: 09/27/2013
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: HUANG, Bo, CHEN, Baohui, QI, Lei
• Granted Patent: US 10,822,606 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 2310/10 Type of nucleic acid
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2310/531 Stem-loop; Hairpin
• CPC Class Codes: C12N 2800/80 Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Y 301/00 Hydrolases acting on ester bonds (3.1)

Abstract

Methods, compositions, and kits are provided herein for CRISPER/Cas-mediated nucleic acid detection or modification.

Claims

1. A small guide RNA molecule comprising from 5′

to 3′

;

a binding region, comprising between about 5 and about 50 nucleotides;

a 5′

hairpin region, comprising;

fewer than four consecutive uracil nucleotides;

or

a length of at least 31 nucleotides; and

a 3′

hairpin region; and

a transcription termination sequence,

wherein the small guide RNA is configured to form a complex with a small guide RNA-mediated nuclease, the complex having increased stability or activity relative to a complex containing a small guide RNA-mediated nuclease and a small guide RNA comprising at least 95% identity to SEQ ID NO;

1 or a complement thereof.

2. The small guide RNA molecule of claim 1, wherein the 5′

hairpin region comprises fewer than four consecutive uracil nucleotides and a length of at least 31 nucleotides.

3. The small guide RNA molecule of claim 1, wherein the small guide RNA molecule further comprises an additional hairpin region designed to interact with a protein or small-molecule to conditionally stabilize the secondary and/or tertiary structure of the small guide RNA molecule.

4. The small guide RNA molecule of claim 1, comprising at least 95% identity to SEQ ID NOs:

2, 3, or 4, or a complement thereof.

5. A composition for nucleic acid modification or detection comprising any of the small guide RNA molecules of claims 1-4, the composition further comprising a small guide RNA-mediated nuclease, wherein the small guide RNA and the small guide RNA-mediated nuclease form a complex having increased stability or activity relative to a complex containing a small guide RNA comprising at least 95% identity to SEQ ID NO:

1 or a complement thereof.

6. The composition of claim 5, wherein the composition is nuclease defective, thereby forming a complex configured to bind to, but not cleave or nick, a target nucleic acid substantially complementary to the binding region of the small guide RNA.

10. The composition of claim 6, wherein the nuclease defective composition comprises a labeled Cas9 protein.

13. The composition of claim 5, wherein the composition has nuclease activity, thereby forming a complex configured to bind and cleave a target nucleic acid sequence substantially complementary to the binding region of the small guide RNA.

14. The composition of claim 13, wherein the small guide RNA-mediated nuclease has nicking activity, but is substantially defective at catalyzing double stranded breaks in the target sequence.

16. An expression cassette comprising a promoter operably linked to a nucleic acid encoding any of the small guide RNAs of claims 1-4.

17. The expression cassette of claim 16, wherein the promoter is an RNA polymerase III promoter.

25. A host cell comprising any one of the small guide RNAs of claims 1-4.

40. A method of modifying a target nucleic acid sequence in a cell, the method comprising:

introducing into the cell;

(a) a small guide RNA of claims 1-4, the small guide RNA specific for the target nucleic acid sequence; and

(b) a small guide RNA-mediated nuclease, thereby forming a small guide RNA;

nuclease complex; and

(ii) incubating the cell to allow the small guide RNA;

nuclease complex to bind to and cleave or nick the target nucleic acid, thereby modifying the target nucleic acid sequence in the cell.

43. The method of claim 40, wherein the method further comprises

(i) introducing into the cell:

(a) a pair of small guide RNAs of claims 1-4, each small guide RNA specific for a target nucleic acid, wherein the pair of small guide RNAs bind to target nucleic acids on a chromosome and flank a nucleic acid region of interest; and

(b) a small guide RNA-mediated nuclease, thereby forming a pair of small guide RNA;

nuclease complexes; and

(ii) incubating the cell to allow the small guide RNA;

nuclease complexes to localize to the target nucleic acid sequence, thereby creating nicks or double stranded breaks that flank the nucleic acid region of interest; and

(iii) incubating the cell to allow non homologous end joining (NHEJ) or homologous DNA repair (HDR) to occur, thereby reducing heterozygosity in the cell or deleting at least a portion of the nucleic acid region of interest.

44. The method of claim 43, wherein the method further comprises introducing into the cell a heterologous nucleic acid that contains regions of substantial homology to the nucleic acid region of interest, thereby incorporating at least a portion of the heterologous nucleic acid into the nucleic acid region of interest.

45. A kit comprising an sgRNA of any one of claims 1-4, and a labeled nuclease defective sgRNA-mediated nuclease.

7-9. -9. (canceled)

11-12. -12. (canceled)

15. (canceled)

18-19. -19. (canceled)

20. An expression cassette comprising a promoter operably linked to a nucleic acid encoding a small guide RNA-mediated nuclease, wherein:

the promoter is a weak mammalian promoter as compared to the human elongation factor 1 promoter (EF1A), wherein the weak mammalian promoter is selected from the group consisting of a ubiquitin C promoter, a phosphoglycerate kinase 1 promoter (PGK), and a TetOn promoter in the absence of an inducer; and

the nucleic acid further encodes a one or two nuclear localization sequences.

21-24. -24. (canceled)

26-29. -29. (canceled)

30. A method of detecting a target nucleic acid sequence in a cell, the method comprising:

introducing into the cell;

(a) one or more small guide RNAs, each small guide RNA specific for the target nucleic acid sequence; and

(b) a labeled nuclease-deficient small guide RNA-mediated nuclease, thereby forming a labeled RNA;

nuclease complex; and

(ii) incubating the cell to allow the labeled RNA;

nuclease complex to localize to the target nucleic acid sequence; and

(iii) detecting the presence, absence, or quantity of labeled complex in the nucleus of the cell, thereby detecting the target nucleic acid.

31. The method of claim 30, the method further comprising introducing at least 5 different small guide RNAs, each specific for a different portion of the target nucleic acid sequence.

32. The method of claim 30, wherein the one or more small guide RNAs are specific for a repeated target sequence.

36. The method of claim 30, wherein the introducing comprises:

forming a complex between;

a first labeled nuclease-deficient small guide RNA-mediated nuclease and one or more small guide RNAs, to form a first labeled complex; and

a second labeled nuclease deficient small guide RNA-mediated nuclease and one or more small guide RNAs, to form a second labeled complex; and

contacting the cell with the first and second complexes.

38. The method of claim 36, wherein each labeled complex is specific for a different target nucleic acid sequence or specific for a different region of a chromosome.

39. The method of claim 36, wherein each labeled complex is labeled with a different label, and the method comprises detecting the presence, absence, or quantity of each labeled complex in the nucleus of the cell.

33-35. -35. (canceled)

37. (canceled)

41-42. -42. (canceled)

46-47. -47. (canceled)